Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo nám. 542/2, CZ-16000 Prague 6, Czech Republic.
J. Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences, Dolejškova 2155/3, CZ-18223 Prague 8, Czech Republic.
Langmuir. 2022 Sep 20;38(37):11284-11295. doi: 10.1021/acs.langmuir.2c01435. Epub 2022 Sep 9.
Adsorption of arginine-rich positively charged peptides onto neutral zwitterionic phosphocholine (PC) bilayers is a key step in the translocation of those potent cell-penetrating peptides into the cell interior. In the past, we have shown both theoretically and experimentally that polyarginines adsorb to the neutral PC-supported lipid bilayers in contrast to polylysines. However, comparing our results with previous studies showed that the results often do not match even at the qualitative level. The adsorption of arginine-rich peptides onto 1-palmitoyl-2-oleoyl--glycero-3-phosphocholine (POPC) may qualitatively depend on the actual experimental conditions where binding experiments have been performed. In this work, we systematically studied the adsorption of R and K peptides onto the POPC bilayer, aided by molecular dynamics (MD) simulations and fluorescence cross-correlation spectroscopy (FCCS) experiments. Using MD simulations, we tested a series of increasing peptide concentrations, in parallel with increasing Na and Ca salt concentrations, showing that the apparent strength of adsorption of R decreases upon the increase of peptide or salt concentration in the system. The key result from the simulations is that the salt concentrations used experimentally can alter the picture of peptide adsorption qualitatively. Using FCCS experiments with fluorescently labeled R and K, we first demonstrated that the binding of R to POPC is tighter by almost 2 orders of magnitude compared to that of K. Finally, upon the addition of an excess of either Na or Ca ions with R, the total fluorescence correlation signal is lost, which implies the unbinding of R from the PC bilayer, in agreement with our predictions from MD simulations.
精氨酸丰富的带正电荷的肽吸附到中性 zwitterionic 磷酸胆碱（PC）双层膜上是这些有效细胞穿透肽进入细胞内部的转位的关键步骤。过去，我们已经从理论和实验两方面证明了多聚精氨酸与多聚赖氨酸相反，吸附到中性 PC 支持的脂质双层膜上。然而，将我们的结果与以前的研究进行比较表明，即使在定性水平上，结果也常常不匹配。富含精氨酸的肽吸附到 1-棕榈酰-2-油酰基-甘油-3-磷酸胆碱（POPC）上可能定性地取决于进行结合实验的实际实验条件。在这项工作中，我们使用分子动力学（MD）模拟和荧光相关光谱（FCCS）实验，系统地研究了 R 和 K 肽在 POPC 双层膜上的吸附。使用 MD 模拟，我们测试了一系列增加的肽浓度，同时增加 Na 和 Ca 盐浓度，表明在系统中增加肽或盐浓度会降低 R 的吸附强度。模拟的关键结果是实验中使用的盐浓度可以定性地改变肽吸附的情况。使用荧光标记的 R 和 K 的 FCCS 实验，我们首先证明 R 与 POPC 的结合比 K 紧密近 2 个数量级。最后，在 R 中加入过量的 Na 或 Ca 离子后，总荧光相关信号丢失，这意味着 R 从 PC 双层膜上解吸，这与我们从 MD 模拟的预测一致。
